Pharmacogenomics

Pharamcogernomics is that aspect where the interesting. facts are brought to the public exclusively.

1. PHARMACOGENOMICS PRESENTED BY : ANAND SAGAR TIWARI M.PHARM (FIRST SEMESTER) DEPT. OF PHARMACOLOGY GUIDED BY : Mrs. ARATI MALPANI M.PHARMA( Ph.D )

2. Overview Basic concepts Gene mapping and cloning of disease gene Genetic variation and its role in health pharmacology Polymorphisms affecting drug metabolism Genetic variation in drug transporters Genetic variation in G protein coupled receptors Application of Proteomics, Genomics, Metabolomics , Functionomics & Nutrigenomics .

3. NOT THIS ONE---

4. Basic concepts : Gene : A gene is the basic physical and functional unit of heredity. Genes are made up of DNA wherein they act as a instruction to make molecules called proteins. All genes don’t code for proteins. The Human Genome Project estimated that humans have between 20,000 – 25,000 genes. Every person has two copies of each gene one inherited from each parent. Though genes are same in most of the people, a small number of them are slightly different. Alleles are forms of the same gene with small differences in their sequence of DNA bases.

5. Human Genome Project It was an ambitious international project that started bin 1990s molecular biologists to understand the genomes of humans and other organisms. It is designed to identify and locate 35,000-40,000 genes present on 23 pairs of chromosome contained in the nucleus of the cell of humans. USA took lead later to be joined in by 14 other countries namely Australia , Canada, Brazil, Denmark, F rance, Germany , Israel, Italy , Japan, Netherlands, Russia, Sweden and UK . It was believed that this project will give insight into the fundamental mechanisms of life and lead to an era of molecular medicine with new ways to prevent, diagnose and treat a disease.

6. Goals of HGP: To determine the complete nucleotide sequence of DNA of each chromosome. To construct the detailed genetic and physical maps of nearly 3 billion base pairs. Detailed study of information about the structure , organization and function of human DNA. To diagnose genetic disease like cancer, cardiovascular disease , autoimmunity and mental illness . To develop efficient technology for the identification of genes. To identify ethical , legal and social implications that may arise from the project.

7. Achievements: Identification of nearly 30,000-35,000 genes . Identification of 3164.7 million nitrogen bases in the human genome. Study of size and number bases of genes. The average gene consists of 3000 bases but the size vary greatly. Establishment of the fact that almost all nucleotide bases are exactly the same in all people (99.9 %). Identification of chromosome I with 2968 genes and Y chromosome with only 231 genes. Identification of 1.4 million locations in human genome where single base DNA difference occurs.

8. Genetic code : The three nucleotide(triplet) base sequences in m RNA that act as a code words fore amino acids in protein constitute the genetic code or simply CODONS . The genetic code may be regarded as a dictionary of nucleotide bases (A,G,C &U) that determine the sequence of amino acid in proteins. These four bases produce 64 different combinations of three base codons. 3 CODONS viz. UAA, UAG, UGA don’t code for amino acids thus known as termination codons or non-sense codons .These CODONS are named as amber , ochre and opal codons. Initiation codons are AUG(methionine) and GUG.

9. World of -OMICS : PROTEOMICS GENOMICS NUTRIGENOMICS METABOLOMICS FUNCTIONOMICS The suffix –OMICS itself means the field of study in B iology. At the same time the suffix –OME is used to address the objects of study of such fields as PROTEOME , GENOME etc.

10. High Throughput Screening High Throughput screening (HTS) is a method for scientific experimentation especially used in drug discovery and relevant to the fields of biology and chemistry. Using robotics , data processing control software , liquid handling devices and sensitive detectors it allows a researcher to quickly conduct millions of chemical , genetic or pharmacological tests. Through this tests one can easily and rapidly identify active compounds , antibodies or genes that modulate a particular bio-molecular pathway. Thus it helps to know the starting point for drug design and for understanding the non-interaction of a particular location.

11. Pharmacogenomics Pharmacogenomics is the study of the role of the genome in drug response. Its name( pharmaco + genomics) reflects in combining of Pharmacology and Genomics . Thus it analyzes how the genetic makeup of an individual affects his/her response to the drugs. It deals with the influence of acquired and inherited genetic variation on drug response in patients by correlating gene expression or single nucleotide polymorphisms with pharmacokinetics and pharmacodynamics. Through the utilization of pharmacogenomics it is hoped that pharmaceutical drug treatments can deviate from what is dubbed as “ONE DOSE FITS ALL”.

12. History : It was recognized by Pythagoras around 510 B.C. when he made a connection between the dangers of fava bean ingestion with hemolytic anemia and oxidative stress .This was later validated to the deficiency of G6PD deficiency. In 1956 reports of prolonged paralysis and fatal reactions linked to genetic variation in patients who lacked butyryl -cholinesterase following administration of succinylcholine injection during anesthesia. In the late 1960s twin studies supported the influence of genetic involvement in drug metabolism with identical twins sharing remarkable similarities to drug response compared to fraternal twins, The term pharmacogenomics first began in 1990s.

13. Gene mapping: Gene mapping is the process of determining the order of and relative distance between genetic markers on a chromosome based on the pattern of inheritance. Gene mapping describes the method used to identify the locus of a gene and the distance in between the genes . The essence of all genome mapping is to place the collection of molecular markers onto their respective positions on the genome. The first step of building a genetic map are the development if genetic markers and a mapping population. The closer the markers are on the chromosome the more likely they are to be passed on to the next generation together. The quality of genetic map depends on the number of genetic markers and the size of the mapping population.

14. Molecular markers are the fragments of DNA that are associated with a certain location within the genome. M olecular markers are used in biotechnology to identify a particular sequence of DNA in a pool of unknown DNA. e.g. RFLP (Restriction Fragment Length Polymorphism);AFLP( Amlified Fragment Length Polymorphism).

15. Types: There are two different types of Mapping Genetic mapping Physical mapping Both maps are collection of genetic markers and gene loci. Genetic maps distances are based on the genetic linkage information while physical maps use actual physical distances usually measured in no of base pairs. Physical map could be a more accurate representation of genome.

16. Uses of gene mapping The great use of genetic mapping is that it can identify the relative positions of genes based solely on their phenotypic effect. Identification of genes is usually the first step in understanding the genome of a species ; mapping of the gene is usually the first step of the identification of the gene . Gene mapping is usually the starting point of many downstream studies. The process to identify a genetic element that is responsible for a disease is also referred to as mapping.

17. Gene cloning Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication in the host cells. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned and the species that will serve as the living host for replication of the DNA. This will generate a population of organisms in which r-DNA molecules are replicated along with the host DNA . Because they contain foreign DNA molecules they can be also called as Transgenic or Genetically Modified Organisms

18. Requirements In standard molecular cloning experiments the cloning of any DNA fragment essentially involves seven steps: Choice of host organism and cloning vector Preparation of vector DNA Preparation of DNA to be cloned Creation of r-DNA Introduction of r-DNA into host organism Selection of organism containing r-DNA Screening for clones with desired DNA inserts and biological properties.

19. Genetic variation Genetic variation is the difference in DNA among individuals. There are multiple sources of genetic variation including mutation and genetic recombination. Identification of genetic variation can be carried out by the variation in the phenotypic characters in either quantitative traits or discrete traits. Genetic variation can be studied by the variation in the order of the bases in the nucleotide of genes.

20. Genetic variation contd ….. Polymorphisms : Variation between individuals in a population Substitution : Fixed variation in between individuals of species. Factors affecting genetic variation are : Mutations Gene flow Genetic drift Natural selection Speciation Reproduction

21. Mutations These are spontaneous and permanent change in the genetic makeup of an individual . This change may produce an alteration in the character. Gene mutation is also called point mutation. Due to mutation in the gene one of the amino acids glutamine gets replaced by valine , resulting in Sickle cell anemia. Again mutation in chromosom e can be leading to either Turner’s syndrome or Klinefelter’s syndrome.

22. Gene flow Gene flow is defined as the transfer of genes between two interbreeding population which differ genetically. Gene flow is mainly brought about by migration in hybridization. When there is a random removal of alleles from a donor population and introduced into a recipient population the allele frequency of both the population is effected. This change in gene frequency leads to increased genetic variation and causes evolutionary change.

23. Genetic drift It is the random change in gene frequency in a small population purely by chance. Genetic drift may lead to the fixation or loss of certain genes irrespective of their adaptive value. Genetic drift is also called FOUNDER EFFECT .

24. Natural selection It is the process of selection of better adapted individuals with useful variations by nature. The fittest or better adapted individuals survive , reproduce and contribute their genes to the next generation while the less adapted are rejected by nature. Thus natural selection is the process of differential reproduction that leads to differential contribution of genotypes to the gene pool of the next generation.

25. Speciation It is the process of formation of new species. During this process organisms with a favorable variations are allowed to reproduce by nature at a faster rate than the others. Due to the cumulative effect of this process new structures are formed which in turns leads to the formation of new species.

26. What is g-protein ? Also known as guanine nucleotide binding proteins . Family of protein that acts as a molecular switch inside the cell. Activity regulated by factors that controls their ability to bind and hydrolyze GTP into GDP . When they are bound to GTP, they are “on” and when they are bound to GDP they are “off”. G-Protein belongs to the larger group of enzymes called GTPase . There are two classes of G Proteins Monomeric small GTPase . Heterotrimeric G- Protein complexes ( alpha,beta and gamma subunit).

27. G Protein coupled receptor 7 trans membrane helices connected by alternating cytosolic extra cellular loop. C terminal : Inside the cell N terminal : Extra cellular region Extra cellular portion has unique messenger binding site Cytosolic loop allow receptor to interact with G Protein The eventual effect of agonist induced activation is a change in the relative orientations of the TM helices (likened to a twisting motion) leading to a wider intercellular surface and revelation of residues of the intracellular helices and TM domains crucial to signal transduction function (i.e. G Protein coupling). In case of inverse agonists or antagonists binding to different sites the effect must be prevention of TM helix orientation.

28. Genetic variation in GPCR G protein coupled receptor is the main molecular target for drug action which are estimated to be having around 600 GPCR genes in the human genome. Confounding factors in assessing the therapeutic relevance of variant GPCR alleles include Interaction of a single drug with multiple closely related receptors. Poorly defined binding pockets to accommodate drug ligands in different orientations or at alternative receptor domains Possibility of multiple receptor conformation Multiple signaling pathways engaged by a single receptor.

29. Causes of gentic variation S equence variations of the human genome S tructure and function of GPCR GPCR coupling to g proteins and other signaling pathways GPCR binding pockets S pontaneous GPCR signaling M ultiple receptor conformations with distinct functions .

30. Causes contd …… Sequence variation introduces variability in the genetic makeup. Suspected to play an important role in diseases and variable response in drug therapy . SNPs account for 80% of sequence variations. Molecular architecture might permit the prediction of functionally relevant domains where sequence variations are more likely to alter receptor function . Normally TM domains are highly conserved, the loops are variable in sequence and length &b the C & N tails represent the most diverse element. Exchange of single amino acid residues can lead to constitutive receptor activation . Considerable no. of human polymorphisms enhance signaling or even the receptor constitutively causing serious genetic disorders.

31. Causes contd …….. 4. GPCRs are flexible structures and may accommodate ligands in various ways. It exists in multiple conformations. Discrete signaling pathways are triggered by discrete conformational states of GPCR. 5. GPCR thought to be coupling to heterotrimeric G Proteins composed of alpha , beta & gamma subunits. It displays considerable heterogenecity with a predicted no. of 27 different alpha, 5 beta and 13 gamma subunits . However proteins like kinases , arrestin and phosphatases modulate receptor functions at different do mains that are possible targets for polymorphic effects.

32. GPCRs CONTD….. SEQUENCE VARIATIONS of GPCRs & ASSOCIATED DISEASES

33. Impaired or Enhanced agonist signaling efficacy : Several inactivating sequence variants of peptide receptors have been associated with congenital disorders. E.g. A point mutation causing truncation of T hyrotropin stimulating hormone receptor leads to. Leydig’s cell hyperplasia. V2 vasopressin receptors : A number of mutations in the gene encoding the V2 vasopressin receptor leads to functionally inactive receptor protein that are causative for nephrogenic diabetes insipidus . This a clear indication that receptor activity depends on intact signaling pathways.

34. Thromboxane A2 Receptor : This receptor performs an essential role in hemostasis by inducing platelet aggregation . An R60L amino acid substitution in the first cytoplasmic loop of TBAX2 receptor causes a dominantly inherited bleeding disorder characterized by defective platelet response to TBXA2. This leads to decreased agonist induced second messenger formation. P2Y12ADP Receptor : This receptor sub-type is shown to be the target for anti thrombotic drugs such as ticlodipine & clopidogrel . 2-nucleotide deletion in a region mapping to the end of TMD6 is associated with a rare bleeding disorder.

35. Importance of genetic variation Genetic variation is important because it allows individuals with a given species to adapt to their environment. We all are born with adaptation if don’t naturally have these differences in our DNA we can’t obtain them. If we are different because of genetic variation and we are able to adapt this is known as Survival of Natural Selection . Adaptation make an organism different and if it occurs over a long enough period of time we get new species.

36. ROLE OF GENETIC VARIATION IN HEALTH Gene therapy equipment G ene addition therapies Gene repairing(editing) therapies V ectors for gene transfer

37. Polymorphisms The existence together of many forms of DNA sequences at a locus within the population. A discontinuous genetic variation that results in different forms or types of individuals among the members of s ingle species. Genetic polymorphisms of drug metabolizing enzyme give rise to distinct subgroups in the population that differ in their ability to perform certain drug transformation reactions. Polymorphisms are generated by mutations in the gene for these enzymes which cause decreased , increased or absent enzyme expression or activity by multiple molecular mechanisms.

38. Contd…. Genetic differences in drug metabolis m are the result of genetically based variation in alleles for genes that code for enzymes responsible for the metabolism of drugs. In polymorphisms the genes contain abnormal base pairs or multiples or abnormal alleles leading to altered enzyme function. Difference in enzyme activity occur at different rates according to racial group.

39. Single nucleotide polymorphisms Single change in one allele of a gene responsible for a variety of metabolic processes including enzymatic metabolism. The combination of alleles encoding the gene determines the activity and effectiveness of the enzyme. The overall function of the enzyme is the phenotype of enzyme function. (Phenotype : The observable physical or biochemical characteristics determined by both genetic makeup and environmental issues.)

40. Drug metabolism The metabolism of drugs and other xenobiotics into more hydrophilic metabolites is essential for their elimination from the body as well as for the termination of their biological effect. Drug metabolism reactions are classified as either Phase-1 functionalization reaction or Phase 2 biosynthetic reactions ( Conjugation reaction). Enzyme system involved with the biotransformation of drugs is located in the liver. These biotransformation reactions are carried out by CYPs (CYTOCHROME P 450 isoforms) and by a variety of transferases . Phase 1 Reactions : Oxidation , Reduction, Hydrolysis. Phase 2 Reactions : Acetylation,Glucouronidation , Sulfation , Methylation

41. Drug metabolising enzymes CYTOCHROME P450 VKORC 1 TPMT

42. VKORC 1 The Vitamin K Epoxide Reductase Complex subunit 1 is responsible for the pharmacodynamics of Warfarin. Along with CYP2C9 are useful for the identification of risk of bleeding during warfarin administration. Warfarin works by inhibiting VKOR which is encoded by the VKORC 1 gene.

43. TPMT Thiopurine methyltransferase catalyzes the S-methylation of thiopurines thus regulating the balance between cytotoxic thioguanine nucleotide and inactive metabolites in hematopoietic cells. TPMT is highly involved in 6-MP metabolism . Excessive levels of 6-MP can cause myelosuppression and myelotoxicity . Myelosuppression : A condition in which bone marrow activity is reduced resulting in fewer red blood cells ,WBC and Platelets.It is a side effect of some cancer treatments.]

44. Cytochrome P450 The most prevalent drug metabolizing enzymes are the Cytochrome P450 enzyme. The term was coined by Omura and Sato in 1962 to describe the membrane bound , heme containing protein characterized by 450 nm spectral peak when complexed with Carbon monoxide. Human CYP families consist of 57 genes , 18 families and 44 subfamilies . CYPs are arranged into these families and subfamilies based on the basis of similarities identified by the amino acid sequence.

45. Nomenclature In case of c ytochromes nomenclature enzymes that share 35-40 % identity are assigned to the same family by an Arabic numeral and those that share 55-70 % make up of a particular subfamily with a designated letter e.g. CYP2D6 refers to family 2, subfamily D and gene number 6.

46. CYPs Contd…. Commonly treated CYPs include –CYP2D6, CYP2C19,CYP2C9,CYP3A4 and CYP3A5. DRUG METABOLISED BY ENZYMES: CYP2C9 : Tolbutamide , Ibuprofen, Diclofenac etc. CYP2C19 : Diazepam, Omeprazole, Propranolol. CYP3A4 : Erythromycin, cyclosporine etc. CYP2D6 : Metoprolol , Codeine etc .

47. Mutations in metabolizing enzymes Polymorphisms affect drug interactions by altering the affect of inhibitors and inducers on the enzyme . It results in an exaggerated effect or minimal effect on the substrate. Inhibitor : An enzyme inhibitor is the one which binds to an enzyme and decreases their activity. Inducer : An enzyme inducer is a type of drug that increases the metabolic activity of an enzyme either by binding to the enzyme either by binding to it and activating it or by increasing the expression of the gene coding for the enzyme.

48. Mutations Contd…… Poor Metabolizers : Two defective alleles (e.g. CYP2D6*4/*5 and CYP2D6*4/*4) or combination of alleles including one resulting in no enzymes (e.g.CYP2DG*5 and CYP2D6*4 deletion). Intermediate Metabolizers : These may be heterozygous having only one wild type allele and one defective allele. Normal Metabolizers : Carry wild type allele (e.g. CYP2D6*1/*3). Wild type alleles encode genes for normal enzyme function. Extensive Metabolizers : Carry one wild type and one amplified gene.( Ex. CYP2D6*1/*2, CYP2D6*A/1 and CYP2D6*1A/*5). Ultrarapid Metabolizers : Carry two or more copies of amplified gene. (e.g. CYP2D6*2/*3)

49. CYP2C9 Enzyme in humans is encoded by CYP2C9 gene. Enzyme being highly polymorphic genetic polymorphism does exist . More than 50 SNPs have been described in the regulatory and coding regions of the CYP2C9 gene with some of them associated with reduced enzyme activity compared with wild type. For CYP2C9 substrates such as Warfarin and Phenytoin , diminished metabolic capacity because of genetic polymorphisms or drug-drug interactions can lead to toxicity at normal therapeutic doses.

50. CYP2C19 It is a member of Cytochrome P450 mixed function oxidase system. It is involved in the metabolism of xenobiotics , including many proton pump inhibitors and anti-epileptics . It is a liver enzyme that acts on at least 10% drugs in current use notably the antiplatelet agent Clopidogrel ( Plavin ) . Also drugs that treat pain associated with ulcers such as omperazole , antiseizure drugs such as mepheytoin , the antimalarial Proguanil and anxiolytic diazepam

51. Polymorphisms Genetic polymorphism do exist for CYP2C19 expression with approx. 3-5% of European and 15-20% of Asian population being poor metabolizers with no CYP2C19 function. In patients with reduced gene activity Clopidogrel may not be metabolized to its active form and therefore may not achieve the required pharmacological activity in the body. Polymorphisms can be observed in CYP2C19*2 , CYP2C19*3 and CYP2C19*17 genes. Clopidogrel being administered as a prodrug it depends on the action of enzyme in the body to observe the desired pharmacological effect.

52. CYP2C19 and Diazepam Diazepam is demethylated by CYP2C19 . Plasma half life is longer in individuals who are homozygous for the effective CYP2C19*2 allele compared to those who are homozygous for the wild type allele. Half life of desmethyldiazepam is also longer in CYP2C19 poor metabolizers. Diazepam induced toxicity may occur as a result of slower metabolism – requires careful dosing in Asian population.

53. CYP2D6 In humans this enzyme is encoded by the CYP2D6 gene. Primarily expressed in liver with expressions in areas of CNS including the S ubstantia nigara . Member of the CYP450 mixed function oxidase system and one of the most important enzymes involved in the metabolism of xenobiotics in the body. Responsible for the metabolism and elimination of approx. 25% of clinically used drugs via the addition or removal of certain functional groups specifically hydroxylation , demethylation and dealkylation . Also activates some prodrugs . Moreover metabolizes endogenous substance such as 5-HT, neurosteroids and both m- tyramine and p- tyramine which are metabolized into dopamine in the brain and liver.

54. Polymorphisms Variation exists in the efficiency and amount of CYP2D6 enzymes produced between individuals. Hence certain will eliminate quickly while for others it is slow(poor metabolizers). Due to genetic polymorphism in it shows the largest phenotypical variability accounting for normal, reduced and non-existent CYP2D6 function in subjects. The genetic basis of CYP2D6 mediated metabolic variability is the CYP2D6 allele located on chromosome 22 . These with certain allelic variation will show increased or decreased or normal or no CYP2D6 function.

55. CYP3A4 It is an important enzyme in the body mainly found in the liver and in the intestine. Oxidizes small foreign organic molecules such as toxins or drugs so that they can be removed from the body. Of the total metabolism carried out by CYP450 about 60% is carried out by CYP3A4. Metabolizes substances like acetaminophen, codeine, diazepam , steroids and certain carcinogens. At the same time many substances are bioactivated by CYP3A4 to form their active compounds and many proteins being toxicated into their toxic forms.

56. Genetic Variability Over 28 SNPs have been reported none of them have significant inter-individual variability in-vivo. Alleles which have been reported to have minimal function compared to wild type include CYP3A4*6 and CYP3A4*17 . Both of these lead to a decreased catalytic activity with certain ligands including testosterone and nifedipin .

57. Genetic variation in drug transporters Introduction : Drug transporters are membrane bound proteins involved in the uptake or efflux of drugs by several tissues such as the intestine , liver, kidneys and brain. They can have a significant impact on the pharmacokinetics of endogenous and exogenous compounds. Drug transporters along with drug metabolizing enzymes are major determinants of drug disposition and are known to alter the response to many commonly used drugs. For delivering efficacious medicine with the right dose for each patient it is important to understand the contribution of genetic variants for drug transporters.

58. Types of transporters Transporters can be divided into two families : Uptake Transporters or Solute linked family [SLC family] Efflux Transporters or ATP binding cassette,[ABC family]. ABC family are expressed in cells of several organs such as liver , small intestine and kidney ,where they influence the pharmacokinetics of drugs. Transporter proteins have wide substrate specificity and therefore influence the pharmacokinetics of the drugs.

59. Solute linked carrier transporters The solute carrier group of membrane transport proteins include 400 members organized into 65 families. Solutes are carried are extremely diverse and include both charged and uncharged organic molecules as well as inorganic ions and the gas ammonia. It contains a number of hydrophobic transmembrane alpha helices connected to each other by hydrophilic intra and extra cellular loops. SLC group includes example of transport proteins that are facilitative transporters ,secondary active transporters, primary active transporters , ion channels and aquaporins .

60. Nomenclature Names of SLC family have the following format SLCnXm where; SLC is the root name( solute carrier ) n is an integer representing a family (e.g. 1-52) X = single letter (A,B,C…) denoting a subfamily M= an integer representing an individual family member. example : SLC1A1 is the first isoform of subfamily of SLC family 1.

61. ATP binding cassette transporter The ABC are a transport system superfamily that is one of the largest and possibly one of the oldest g ene families. Consists of multiple subunits one or two of which are transmembrane subunits and one or two of which are membrane associated with AAA ATPases . The ATPase subunits utilize the energy of ATP binding and hydrolysis to provide the energy needed for the translocation of substrates across membranes either for uptake or for export of the substrate. ABC uptake porters take up a large variety of primary and secondary metabolites. Some of them are involved in tumor resistance , cystic fibrosis and a range of other inherited human disease.

62. ABC genes are essential for many processes in the cell and mutations in human genes cause or contribute to several human genetic diseases. Many of them have been characterized and shown to be casually related diseases present in humans as Cystic fibrosis , Adrenoleukodystrophy , Stargardt disease, Ataxia and Hyperinsulimenic hypoglycemia. Most of the eukaryotic ABC transporters are effluxers some are not directly involved in transporting substance. Human ABC transporters are involved in several diseases that arise from polymorphisms in ABC genes and rarely due to complete loss of function of single ABC proteins.

63. P- Glycoprotein P –glycoprotein also known as multidrug resistance protein 1 or cluster of differentiation 243 is an important protein of the ell membrane that pumps many foreign substances out of the cells. It is an ATP dependent efflux pump. Discovered in 1971 by Victor Ling . The glycoprotein is encoded by ABCB1 gene. It transports various substrates across the cell membrane including drugs like Colchicine, Quinidine, Tyrosine kinase inhibitors( Gefitinib , Sunitinib ).

64. ABC transporters subfamilies There are 49 known ABC transporters present in humans which are classified into 7 families by Human Genome Organization. ABCA ---- Family contains some of the largest transporters. 5 of them are located in a cluster in the 17q24 chromosome . Responsible for the transportation of cholesterol and lipids. e.g. ABCA1,ABCA12 . ABCB ---- Consists of four full and seven half transporters . Some are located in the BBB, liver , mitochondria which transport peptides and bile. e .g. ABCB5 ABCC ---- Consists of 12 full transporters . Used in ion transport, cell surface receptors, toxin secretion. Includes CFTR protein which causes cystic fibrosis when deficient. e.g. ABCC6

65. Subfamilies contd ….. 4) ABCD ---- Consists of 4 half transporters. All are used in peroxisomes. e.g. ABCD1 . 5) ABCE/ABCEF ---- Consist of 1 ABCE & 3 ABCF proteins . These are not actually transporters but merely ATP binding domains that were divided from the ABC family , but without the transmembrane domains. These proteins regulate protein synthesis or expression. e .g. ASBCE1, ABCF1,ABCF2 . 6) ABCG ---- Consists of 6 reverse half transporters with the NBF at the NH3+ end and the TM at the COO- end . Transports lipids, diverse drug substrates, bile cholesterol and other steroids. e.g. ABCG2, ABCG1 .

66. Genetic variation ABCA1 group is implicated in the development of genetic diseases . e.g. In reverse Tangier’s disease the ABCA1 protein is mutated. Also the ABCA4 maps to a region of chromosome p21 that contains the gene for Stargardt’s disease . In rod photoreceptors the gene is highly expressed and is mutated in Stargardt’s disease. For ABCB group cells that overexpress this protein exhibit multi-drug resistance . ABCE group is composed of genes that have ATP binding domains that are closely related to other ABC transporters but these genes don’t encode for transmembrane domains.

67. 5) ABCG genes have orientation opposite to that of other ABC genes. The ABCG2 gene was discovered in cell lines selected for high level resistance for M itoxantrone and no expression of ABCB1 or ABCC1 or ABCG2 can export A nthrocycline anticancer drugs as well as T opotecan as substrates. 6) Mutation in ABCD1 is responsible for Adrenoleukodystrophy . The cells of ALD patient feature accumulation of unbranched saturated fatty acids. 7) ABCC are also known as multidrug resistance proteins[MRPs ]. Disease like cystic fibrosis can be observed when there is genetic mutation in CFTR ( cystic fibrosis transmembrane regulator) a part of this subfamily. Mutations in Sulphonylurea , a part of this subfamily receptors are a potential cause of Neonatal diabetes mellitus.

68. Cystic fibrosis A genetic disorder that affects mostly the lungs but also the pancreas, liver, kidneys and intestine. It is inherited in autosomal recessive manner and caused due to the presence of mutations in both the copies of the gene for the CFTR Protein. Those with a single working copy are carriers and otherwise mostly healthy. CFTR is involved in the production of sweat, digestive fluids and mucus. Most common among North European and affects one out of every 3000 newborns .

69. Adrenoleukodystrophy This a disease linked to the X chromosome( X- Linked recessive inheritance). It is a result of fatty acid buildup caused by the relevant enzymes not functioning properly, which then causes damage to the myelin sheath of the nerves, resulting in seizures and hyperactivity. It is caused by mutations in the ABCD1 gene located on the X chromosome that codes for ALD a peroxisomal membrane transporter protein. Males with an ABCD1 mutation are hemizygous as they have only a single X chromosome. Female carriers will avoid severe manifestations but later may be symptomatic. Detection of mutation identifies an individual who is affected there is no genotype-phenotype correlation.

70. Stargardt disease It is the most common inherited single-gene retinal disease. It usually has an autosomal recessive inheritance caused by mutations in the ABCA4 gene. Symptoms commonly include wavy vision, blind spots, blurriness,, loss of depth perception. The carrier frequency in the general population of ABCA4 alleles is 5-10% . Different combinations of ABCA4 genes will result in widely different age of onset and retinal pathology.

71. - Omics world explored Genomics : Genomics is that field of biology that focuses on the structure, function , evolution ,mapping and editing of genes. The field also involves the study of intragenomic (within the genome ) phenomena such as epistasis(effect of one gene on another), pleiotropy ( one gene affecting more than one trait) , heterosis ( hybrid vigor ) and other interactions in between loci and alleles within the genome.

72. Genomics : In contrast to genetics which refers to the study of individual genes and their roles in inheritance. Genomics aims at the collective characterization and quantification of all of an organism’s genes, their interrelations and influence on an organism. Genomics also involves the sequencing and analysis of genomes through high throughput DNA sequencing and Bioinformatics to analyze the function of each genome.

73. Applications Genomics has provided applications in many fields namely ; Genomic medicine : Next generation genomic techniques has allowed researchers to drastically understand the genetic bases of drug response and disease. Synthetic biology and bioengineering Conservation genomics : Conservationist can use the information gathered by genome sequencing in order to better evaluate genetic factors key to species conservation such as the genetic diversity of a population or whether an individual is heterozygous for a recessive disorder.

74. Proteomics The word was coined in the year 1997. Proteomics is defined as the large scale study of proteins. As known proteins are vital parts of living organisms with many functions. Proteome is the entire set of proteins. Proteome gets modified as an individual undergoes stresses or with time or distinct requirements. Involves the overall exploration of Proteomes from the overall level of protein composition, structure and activity.

75. Types of Proteomics Interaction proteomics : Protein-protein association Expression proteomics : Protein quantification Level of any protein in cell at any given time can be controlled by : Rate of transcription of the gene. The efficiency of the translation of m-RNA into protein. The rate of degradation of protein into cell.

76. Applications: Mining Protein expression profiling : identification of proteins in a sample as a function of a particular state of cell. Protein network mapping : To determine protein interaction with each other in living organism. Mapping of protein modifications : Task to identify how and where proteins are modified post- translationally . Helps to study drug MoA , Disease Biomarkers ,Epigenetics, Spatial localization etc.

77. Nutrigenomics Nutrigenomics or nutrition genomics is a science studying the relationship between human genome ,nutrition and health. People in this field work towards developing an understanding of how the whole body responds to a food via systems biology. [ Systems biology is the computational and mathematical analysis and modeling of complex biological system. The Human Genome Project is an example of applied systems thinking in biology which has led to new collaborative ways of working on problems in the biological field of genetics.]

78. The four basic tenets of Nutrigenomics are : Improper diets are risk factor for disease. Dietary chemicals alter gene expression or change gene structure. The degree to which diet influences the balance between healthy and disease states may depend on individuals genetic make up. Some diet regulated genes are likely to play a role in the onset , incidence, progression or severity of chronic disease.

79. Advantages Increased focus on a healthy diet and lifestyle. Increased awareness of risk of certain conditions. Improved health quantity of life. Focus on prevention of diseases. Decrease morbidity and premature mortality. Reduced healthcare concepts. Better understanding of the mechanisms involved in the disease suspectibility .

80. Disadvantages Attention is drawn away from other modifiable risk factors. Focus only on specific nutrients and foods. Increased costs associated with personalized diets and designer foods. Sometimes plagued with misleading claims.

81. Specific dietary profiles can modulate the delicate balance between health and disease acting either directly or indirectly or gene expression. Personalized diets which takes into account individual genotype represents the ultimate goal of NUTRIGENOMICS . Certain foods like cauliflower, broccoli contain chemicals that actually tell our gene to direct biosynthesis of these enzymes. In individuals who lack the required gene or possess gene which give unclear instruction for making an enzyme that metabolites amino acid – Phenylalanine leading to amino acid build up causing brain damage .

82. Applications Nutritional science originally emerged as a field that studied individuals lacking certain nutrients and the subsequent effects such as scurvy which results from lack of Vitamin C. Obesity is another aspect studied. It’s genetic variation that individuals respond to diet differently. By exploring the interaction between dietary pattern and genetic factors one can suggest the field that could prevent or reduce obesity. Ex. Prader-Willi Syndrome [Characterized by insatiable appetite where the paternal copy of the chromosome is erroneously deleted and the maternal loci is inactivated by over methylation.] The APO B SNP rs5125356 is another diet related variation ; the A/G heterozygous genotype was found to have association with obesity in terms of BMI and waist circumference.

83. Future aspects In the coming years NUTRIOGENOMICS has the promise to be the most interesting and exciting fields for cutting edge research. The different – OMICS discipline offer a variety of powerful ways to understand what is going inside the cells in response to nutrients and to see how these responses differ from person to person. Over the past decade a lot of study has been carried out regarding which antioxidant is found in which food and at which level. It all started in 1920 when Goldberger showed that he could cure pellagra by simply adding Baker’s yeast to the diet . After another 10 years it was concluded that it was Niacin in bakers yeast that cured it.

84. Functionomics Functionomics or functional genomics is a field of molecular biology that attempts to describe gene functions and interactions. Functinomics make use of the vast data generated by proteomic and transcriptomic projects . Functional genomics focuses on the dynamic aspects such as gene transcription, translation, regulation of gene expression and protein-protein interactions. A major challenge remains to integrate functionomics with genomics and metabolomics data and mainly their integration with their physiological and pathophysiological functional interpretation.

85. Applications The main goal of functional genomics is to understand the function of genes or proteins eventually all components of genome. It involves the study of mutation and polymorphism (SNP) as swell as the measurement of molecular activities. Functionomics is concerned with Gene Interaction mapping. To study the DNA/protein interactions. Protein formed by the translation of m-RNA play a major role in regulating gene expression and to understand that it is necessary to identify DNA sequence that interact with. Required for DNA accessibility assays . These assays include Chip sequencing , CUT& RUN sequencing and Calling cards .

86. -OMICS chain DNA ----------- GENOMICS | RNA ------------ TRANSCRIPTOMICS | PROTEINS --------- PROTEOMICS | BIOCHEMICAL ---------- METABOLOMICS | BIOLOGICAL PHENOTYPE

87. Metabolomics Definition : Metabolomics is the scientific study of chemical processes involving metabolites , the small molecule substrates, intermediates and products of metabolism. The metabolome represents the complete set of metabolites in a biological cell , tissue or an organ or an organism. Thus metabolomics provides a direct functional readout of the physiological state of an organism.

88. Do you know ?? Genome can tell what could happen. | T ranscriptome can tell what appears to be happening | Proteome can tell what makes it happen | M etabolome can tell what has happened and what is happening.

89. How it started ? The first metabolite database called METLIN for searching fragmentation from tandem mass spectrometry experiments was developed by the Sizudak Lab at the SCRIPPS Research Institute in 2005. In January 2007 scientists at the University of Alberta and the University of Calgary the first draft of the Human Metabolome . Thus concluded that each type of cell and tissue has a unique metabolic fingerprint that can elucidate organ or tissue specific information.

90. Metabolites Metabolites are substrates , intermediates and products of metabolism. Within the context of metabolomics a metabolite is usually defined as any molecule less than 1.5 kDa in size. Exometabolomics or Metabolomic Fingerprinting is the study of extracellular metabolites. It uses many techniques from other subfields of metabolomics with application in biofuel development, bioprocessing, determining drug mechanism of action and studying intercellular interactions.

91. Applications : P harmacology and pre-clinical drug trials N ew born screening T oxicology T ransplant monitoring T ools for functional genomics A pplied in novel therapeutics and detection of various forms of cancer. Metabologenomics is a novel approach to integrate metabolomics and genomics data by correlating microbial exported metabolites with predicted biosynthetic genes. Fluoxomics is the further development of metabolomics which helps to determine the reactions and can trace metabolites in a biological system over time.

92. Basic workflow : Sample collection,treatment , processing-------- Separation technique[GC,HPLC,CE] ------- Detection Technique( NMR, MS)-------- Data analysis using multivariate analysis[ PCA( Principle Component Analysis)]---------- Validation followed by clinical application -------- Sample collection, treatment and processing. Human Metabolome Project : On 23 rd January ,2007 , Human Metabolome Project led by Dr.David Wishart of the University of Alberta, Canada completed first draft of Human Metabolome consisting of database of approx. 2500 metabolites .

93. Problems and challenges : Metabolites are having wide range of molecular weight and large variation in concentration. Metabolome is much more dynamic than proteome and genome, thus more time sensitive. Number of metabolites existing are far less than the no of transcripts. Not all metabolites can be identified.

94. Future aspects: If Pathognomonic metabolic profiles of various diseases can be identified and validated in various body fluids metabolomics may save time , cost and effort in obtaining definitive diagnosis in situations where no other test can provide answer. Can be adopted as a minimally invasive tool. Improved sensitivity will also be positive using cryogenecially cooled NMR probes known as Cryoprobes . Analysis of metabolites in known stress response pathways by molecular profiling helps to response to environmental or genetic perturbations. Thus a multi-metabolite profile obtained from high resolution MS(Mass Spectroscopy) offers potential for identification of robust biomarkers to predict radiation toxicity of organs.

95. Pharamcogrenomics [ Applications ] Improved drug safety and reduced ADRs. Tailor treatments to meet patients unique genetic pre-disposition, identifying optimal dosing. Improved drug discovery targeted to human disease Improved proof of principle for efficacy trials May be applied to the areas of Pain management, Cardiology, Oncology, Psychiatry etc. In Cancer treatment pharmacogenomics tests are used to identify which patients are most likely to respond to certain types of drugs.

96. Challenges Although there appears to be a general acceptance of the basic tenets of Pharmacogenomics amongst physicians and healthcare professionals there are some challenges to be addressed. Limitation on how to apply the test into clinical practice and treatment. Lack of availability of test. Understanding and interpretation of evidence based research. Ethical, Logical and Social issue. No sufficient evidence to validate the cost-effectiveness and cost-consequence of the test.

97. Future aspects Computational advances have enabled cheaper and faster sequencing .Research has focused more on the genomic mining and –OMIC technologies. Also as the cost decreases the development of personalized drug therapies will increase. Technology now allows for the genetic analysis of hundreds of target genes involved in medication and response in less than 24 hours for under 1000 dollars(1000$).

98. Pharmacogenomics in India The current definition of PRECISION MEDICINE – Understanding disease at a deeper level in order to develop more targeted therapy clearly requires the armament of pharmacogenomics to succeed. Some of them are Neo-adjuvant chemotherapy[NACT] or research on P-glycoproteins. NACT is usually provided to patients with locally advanced breast cancers. P- gp an ATP dependent efflux pump is used as a part of NACT for the management of cancer. ABCB1 gene codes for the P- gp . Case study in 48 patients showed that those with high expression of P- gp had 3.6 times more chance of having poor response to NACT.

99. Conclusion: Though the concept of Pharmacogenomics is new it has the potential in it to be adopted as the pioneer to provide Tailor-made therapy to the patients thereby bringing the concept of PRECISION MEDICINE a reality.

100. Reference I nternet R ang and Dale molecular P harmacology G enetic variation in human G protein coupled receptors: Implications for drug therapy; W olfgang S adee;Elen H oeg ; J ulie L ucas and D arwin W ang.